Adenoviruses (Ad) are major causative agents of respiratory, ocular and gastrointestinal diseases. Replication-defective and conditionally replicating Ad vectors are also being employed in a significant number of human gene transfer trials as well as for the development of anti-microbial vaccines. Our proposed studies capitalize on the recent progress made and technology/expertise obtained in determining the first crystal structure of human adenovirus at 3.5 resolution. Even though significant structural insights were gained, a number of notable differences were found between the refined x-ray maps and the high-resolution cryoEM structures of the major capsid protein (hexon) as well as accessory proteins. Moreover, we still lack detailed knowledge of the location of the key cement protein, PVI, implicated in membrane penetration during cell entry. We propose to resolve the discrepancies with regards to the structure and identities of cement proteins, elucidate the critical protein-protein interactions that stabilize the Ad capsid by obtaining a refined model of HAdV, using heavy atom labeling of Met/Cys residues and/or by docking into the virus electron density maps the modular domains of accessory proteins, determined independently at high resolution. In addition, we will evaluate structure-function relationships of the altered Ad capsid by performing mutagenesis and infectivity studies. Specifically, this proposal will accomplish the above goals by: 1) accurately identifying the accessory proteins and the associated protein-protein interactions that stabilize the native AdV capsid and evaluate the conditions that affect the stability of the vertex region; 2) analyzing the structure-function relationships of the deletin mutants of cement proteins and the effectiveness of the modified Ad capsids in vector-based gene delivery and 3) determining the structure of immature AdV particles that represent an early stage of adenovirus assembly. These investigations and subsequent comparative analysis of their structures should provide greater understanding of the interactions that stabilize the adenovirus particle and structure-function relationships of the altered Ad types, which possess unique biophysical and biochemical characteristics.